Calcium plays a central role as a second messenger in photoreceptor transduction. The modulation of sensitivity during light and dark adaptation does not occur if Ca2+ is prevented from changing in the outer segment, and the change in Ca2+ concentration is thought to produce Ca2+-dependent changes in the rate of guanylyl cyclase, rhodopsin gain and lifetime, and cGMP-gated channel opening. Ca2+ may also play a central role in photoreceptor death during retinal degeneration. Since the regulation of the free Ca2+ concentration is vitally important to the normal physiology of the photoreceptor and may also be important in the etiology of degeneration, the measurement of free Ca2+ is crucial for our understanding of the cell biology of this important sensory receptor. The investigator has, therefore, developed a novel technique, called spot confocal microscopy, to determine the level of Ca2+ in darkness and to make quantitative measurements of the changes in free Ca2+ concentration produced by stimulation. This technique is considerably more flexible and sensitive than previous methods and has been used to make accurate measurements of Ca2+ not only in amphibian rods and cones but also in the photoreceptors of mammals such as mice. In this application, the investigator is proposing to use electrophysiology and spot-confocal microscopy to answer two questions of fundamental importance to the life and death of photoreceptors. First, to address how light changes the free Ca2+ concentration in amphibian photoreceptors, a variety of techniques will be used to investigate light-dependent Ca2+ release and re-uptake from salamander rods and cones. Second, to address how light changes the free Ca2+ in other organisms, particularly mammals, measurements from transgenic animals will be used to probe the mechanism of light-dependent Ca2+ release and re-uptake, and to explore a possible correlation between the Ca2+ concentration and the initiation of apoptosis and cell death.